Expansion of human hepatocytes and their application in three-dimensional culture and genetic manipulation

  • Nat Protoc. 2025 Dec;20(12):3722-3754. doi: 10.1038/s41596-025-01211-2.
Kun Zhang  #  1  2 ,  Xiang Yuan  #  3 ,  Shuang Lu  3 ,  Yajing Shu  3 ,  Chenhua Wang  3 ,  Jin Cen  3 ,  Baihua Wu  3 ,  Lijian Hui  4  5  6
Affiliations
  • 1. Key Laboratory of Multi-Cell Systems, Shanghai Institute of Biochemistry and Cell Biology, Center for Excellence in Molecular Cell Science, Chinese Academy of Sciences, Shanghai, China. [email protected].
  • 2. Department of Histoembryology, Genetics and Developmental Biology, Shanghai Key Laboratory of Reproductive Medicine, Shanghai Jiao Tong University School of Medicine, Shanghai, China. [email protected].
  • 3. Key Laboratory of Multi-Cell Systems, Shanghai Institute of Biochemistry and Cell Biology, Center for Excellence in Molecular Cell Science, Chinese Academy of Sciences, Shanghai, China.
  • 4. Key Laboratory of Multi-Cell Systems, Shanghai Institute of Biochemistry and Cell Biology, Center for Excellence in Molecular Cell Science, Chinese Academy of Sciences, Shanghai, China. [email protected].
  • 5. School of Life Sciences, University of Chinese Academy of Sciences, Beijing, China. [email protected].
  • 6. School of Life Science and Technology, ShanghaiTech University, Shanghai, China. [email protected].
  • # Contributed equally.
PMID: 40715744 DOI: 10.1038/s41596-025-01211-2
Abstract

Hepatocytes are one of the most important cell types in the liver, carrying out key functions. They are essential for hepatocyte-based therapy, disease modeling and drug development. However, the availability of primary human hepatocytes (PHHs) is limited by a shortage of donors. It is therefore of great value to expand PHHs in large quantities. Here we provide a detailed protocol for the large-scale expansion of PHHs (proliferating human hepatocytes, ProliHHs) derived from healthy donors and patients with inherited liver diseases, which can be rematured in a three-dimensional culture system. Moreover, we provide a protocol for the genetic manipulation of ProliHHs, including lentivirus transduction and CRISPR-Cas9-mediated knockout and knock-in. The protocol described here will help to realize the full potential of ProliHH-based therapy, organoid-based liver disease modeling and drug screening. The protocol to expand PHHs takes ~1-2 months, the protocol to establish the 3D-cultured ProliHHs takes ~8 d and the protocol to perform gene editing takes ~3 d. Personnel with basic scientific training can conduct these protocols.

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